Breaking the crossability barriers between disomic tetraploid Solanum acaule and tetrasomic tetraploid S. tuberosum

Summary A combination of compatible second pollinations and embryo rescue was applied for systematic production of true tetraploid hybrids from crosses between disomic tetraploid Solanum acaule and tetrasomic tetraploid potato, S. tuberosum. Several genotypes of tetraploid potatoes were pollinated with S. acaule, and the compatible second pollinations were made on the following day, with a genotype of S. phureja, IvP 35 to promote fruit development. Embryo rescue was carried out in 21 families, 14 to 27 days after the first pollination. A total of eight plants were obtained from the embryo rescue and their chromosome numbers were counted in the root tips. Three of the eight plants were identified as tetraploid, and five others as diploid. Morphology, isozyme banding patterns, and pollen stainability, as well as potato spindle tuber viroid (PSTVd) resistance, indicated the hybrid nature of the three plants. This is the first report of successful tetraploid hybrid production between disomic tetraploid S. acaule (4x) and tetrasomic tetraploid potatoes. Seed set from the crosses between one of hybrids and diploid potatoes indicated workable levels of both male and female fertility for introgression of valuable genes from S. acaule into the cultivated potato gene pool. The methodology used may be applied to other disomic tetraploid tuber-bearing Solanum species and with some modifications also to distantly related solanaceous species and genera.     

Barriers to hybridization of Solanum bulbocastanum Dun. and S. Verrucosum Schlechtd. and structural hybridity in their F1 plants

Summary Results of reciprocal crosses between Solanum verrucosum (2n=2x=24) and S. bulbocastanum (2n=2x=24) are described in terms of pollen tube behaviour in styles, of berry and seed set, of fertility and of meiotic behaviour of the F₁ hybrids. Pollen tube growth of S. verrucosum is strongly inhibited in styles of S. bulbocastanum, whereas no inhibition is observed in the reciprocal cross. Therefore S. bulbocastanum x S. verrucosum fails to set berries or seeds, whereas the reciprocal cross produces both berries (54.4% berry set) and seeds (0.3 per berry). Only 14.6% of the seeds germinate. Both the diploid and corresponding tetraploid hybrid plants are vigorous, flower abundantly, have a rather regular meiosis (mainly rod bivalents), but show a high degree of cytoplasmic-genic male sterility. Crossability of the diploid hybrid plants is nil when used as pollen parents and near to zero when used as pistillate parents. The barriers to hybridization of the parent species (unilateral inhibition of pollen tube growth, somatoplastic sterility, cytoplasmic-genic male sterility and structural differences of the parental chromosomes) are discussed and methods are suggested to overcome these barriers.     

The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum

Summary To produce hexaploid (or other polyploid) hybrids, diploid or tetraploid Hordeum vulgare was crossed with hexaploid or octoploid H. bulbosum, and perennial triploid hybrids between the two species were treated with colchicine. The crosses did not yield viable plants: seedset was low, the seed aborted and embryo culture was unsuccessful. The colchicine treatments geve rise to plants in which hexaploid chromosome numbers were observed. At the hexaploid level chromosomal instability occurred, resulting in chromosome elimination.The colchicine-treated triploid hybrids showed in the first years after the treatment better fertility after open flowering than untreated plants, but the level of fertility remained very low. The offspring consisted of haploid, diploid and approximately triploid plants like H. vulgare, tetraploid and approximately tetraploid plants like H. bulbosum, and plants with hybrid morphology and unstable chromosome number, which were highly sterile. Thus the crossing barrier between H. vulgare and H. bulbosum could not be broken down at higher ploidy level.     

Barriers to interspecific hybridization in Ulmus americana

Summary Crossability barriers between the tetraploid Ulmus americana and five other diploid elm species were investigated using both fluorescence and scanning electron microscopy. Both pollen germination and pollen tube development were inhibited in all interspecific crosses. Pollen tube behavior on the stigma of U. americana was unchanged when pollen of a colchicine-induced U. pumila tetraploid was substituted for diploid U. pumila pollen.Self-incompatibility was observed in one of two American elms studied and manifested itself via reduced pollen germination and inhibited pollen tube growth on the stigma surface.The results indicate that crossing barrier(s) between U. americana and other elm species occur at the stigma surface and operate independently of ploidy level. This conclusion is discussed in relation to current breeding efforts to transfer Dutch elm disease resistance into the American elm via interspecific hybridization with Eurasian elms.     

Identification of barriers to interspecific crosses in the genus Trifolium

Summary A study of pre- and post-fertilisation barriers after interspecific crosses of diploid and tetraploid Trifolium pratense L. and wild species T. alpestre L., T. medium L. and T. sarosiense Hazsl. was aimed at finding of a promising cross combination for obtaining hybrids. The growth of pollen tubes was arrested in interspecific crosses mainly when T. pratense was at a diploid level. To investigate the post-fertilisation barriers in detail, the hybrid embryo viability was traced by two clearing treatments of immature seeds: (1) using chloral hydrate (which proved to be most appropriate); and (2) a mixture of benzyl benzoate and dibutly phthalate. In interspecific combinations T. pratense (4×) × either T. alpestre or T. sarosiense, enlargement of immature seeds occurred, but no hybrid embryo was traced. Of the wild species used as a male parent for crosses, T. medium was the only exception from the point of view of fertilisation. Globular, heart and the early torpedo stages of hybrid embryos were observed 7 days after pollination (DAP) but only when T. pratense was at a tetraploid level. When T. pratense (2×, 4×) was used as a male parent for interspecific crosses with T. alpestre, T. medium and T. sarosiense, strong defects in various stages of embryogenesis were observed, particularly wrinkled and narrowing embryo sacs caused by an expansion of endothelial cells. We conclude with the following finding: (1) to make crosses only in one direction with T. pratense as a female parent and T. medium as a male; (2) to use tetraploid plants of T. pratense; (3) and to excise hybrid embryos at an early torpedo stage, about 7 DAP.     

Polyploidy and sexual polyploidization in the genus Vaccinium

Genus Vaccinium,consisting of blueberries, cranberries, lingonberries and many related wild species, includes diploid, tetraploid, and hexaploid species. Most evidence indicates that the tetraploid species are autotetraploids, with non-preferential bivalent chromosome pairing. Although homoploid interspecific crosses usually produce numerous fertile hybrids if the parents are from the same section of the genus, inter sectional crosses at the diploid level normally produce no seedlings, weak seedlings, or seedlings that are very low in fertility. There is a strong but not complete triploid block within Vaccinium. Even with insections, tetraploid × diploid (and the reciprocal) crosses normally give only a few tetraploid and a few triploid hybrids. Hexaploid × diploid crosses within sections are very hard to make, and the few hybrids that have been obtained are pentaploid. The frequency of 2n gametes varies,both among genotypes within species and among species. Vaccinium pollen is normally shed in tetrads, and the frequency of large pollen grains shed in dyads can be used to estimate 2n gamete frequency. Cultivated blueberries occur at both the tetraploid and the hexaploid levels, and there are important genetic resources in the diploids. Unreduced gamete production has permitted transfer of genetic material from the diploid to the tetraploid level and from the diploids and tetraploids to the hexaploid level via triploid hybrids. Intersectional crosses can occasionally produce tetraploid Vaccinium hybrids that appear to behave as amphidiploids and have medium to high fertility. CommercialVaccinium cultivars are normally propagated by cuttings. Intersectional hybridization, chromosome doubling, and asexual propagation could permit the production of novel hybrid combinations with value as ornamentals or in fruit production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Studies of pollen grain germination,pollen tube growth,micropylar penetration and seed set in intraspecific and intergeneric crosses within three Cruciferae species

Summary Pollen grain germination, pollen tube growth and micropylar penetration were investigated in intraspecific and intergeneric crosses involving Brassica napus L. ssp. oleifera (Metzg.) Sinsk. (oil-seed rape or fodder rape), xBrassicoraphanus Sageret (Raparadish) and diploid (2x) and tetraploid (4x) accessions of Sinapis alba L.(white mustard). For the reciprocal intergeneric crosses between B. napus and xBrassicoraphanus no effective barriers to pollen tube growth on stigmata or in styles were observed. The resulting low frequency of hybrid plants was mainly associated with a low rate of ovules with micropylar penetration per siliqua or with embryo abortion. Hybrid plants could be obtained without use of embryo rescue. In reciprocal crosses between B. napus and S. alba 2x or 4x incongruity barriers were observed on the stigma, in the style, and in the ovary resulting in a low frequency of ovules with micropylar penetration per siliqua. Open flower-pollination compared to bud-pollination generally was the more favourable procedure for pollen grain germination and pollen tube growth in crosses involving S. alba, but for micropylar penetration and seed set no differences were observed. Crosses between S. alba 2x () and B.napus () were found to result in a higher frequency of ovules with micropylar penetration as compared to reciprocal crosses or crosses with S. alba 4x. All reciprocal crosses between B. napus and S. alba 2x or 4x were unsuccessful when no embryo rescue was applied. Embryo rescue shortly after pollination, i.e. 2 to 5 days, however, resulted in hybrid seeds and plants, but only when applied to crosses between S. alba 2x () and B. napus (). The possible effects of the genome constitution, taxonomic distance and the parthenogenetic and parthenogenesis inducing ability of the parental genotypes on the observed malfunctions at the pre-and/or post-zygotic stage of the pollen grain-pistil interactions are discussed.Abbreviations DAP Days After Pollination - IAA Indole-3-Acetic Acid - kin Kinetin     

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape. This revised version was published online in August 2006 with corrections to the Cover Date.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

Cross compatibility between diploid and tetraploid perennial ryegrass (Lolium perenne L.)

Summary A number of crosses were made between diploid and tetraploid Lolium perenne plants in order to determine the degree of cross compatibility between the two different ploidy levels. The range of hybridization undertaken involved diploidxdiploid, and tetraploid x tetraploid control crossings, diploid x tetraploid crosses, tetraploid x diploid crosses and mixed pollinations. The seed set, the germination capacity of resultant hybrid seeds, and the chromosome constitution of derived seedlings was determined. In addition attempts were made to culture 12 day-old hybrid embryos from the diploid x tetraploid and reciprocal crosses.The crossing results obtained indicated no barrier to fertilization between diploids and tetraploids, but the irregular and poor development of the seed accompanied by much reduced germination, indicated unsatisfactory endosperm establishment of large numbers of triploids, as confirmed by the result of culturing embryos dissected from 12-day old seeds.In preliminary studies undertaken to determine the extent to which diploid and tetraploid ryegrass varieties intercross when grown in close proximity for seed production, Griffiths and Pegler (1966) observed a very low incidence of triploids amongst the progenies of diploid plants exposed to the pollen of tetraploid plants and also amongst the progenies of tetraploid material exposed to the pollen of diploid plants.In these and subsequent studies it was noted that only a very small proportion of the so-called fully formed seeds derived from diploid x tetraploid crosses did actually germinate. When, in 1964, propagules of clones of S.24 perennial ryegrass, well separated from one another, were placed in drills of the tetraploid perennial ryegrass variety Reveille, the proportion of florets which formed caryopses, and of caryopses which germinated was as follows (Table 1).The data (Table 1) clearly indicate that a considerable proportion of the S.24 florets had been fertilized by 2n pollen and had formed caryopses, but most of these had proved to be defective. As noted in the previous studies, the frequency of triploid seedlings was low, thus suggesting incompatibility between the n and 2n gametes for the production of viable zygotes.Of the total progenies classed as tetraploids in the early seedling stage, 80% proved to be ryegrass x tall fescue F₁ hybrids. These arose as a result of free crossing with     

Inheritance of sesquiterpene lactone types within the Achillea millefolium complex (Compositae)*

Successful crossing experiments between tetraploid individuals of Achillea ceretanica, A. collina, A. distans ssp. styriaca, and A. pratensis (A. millefolium complex, Compositae) were carried out. Sesquiterpenes of 1013 hybrid plants obtained from 13 crossings were studied by qualitative thin layer chromatography (TLC). F₁-progenies from parent plants with guaianolides (GU-type), as well as the offspring of a back-cross, uniformly contained proazulenes. Diallel crosses between plants exclusively with eudesmanolides (EU-type) resulted in a 7:1 segregation-ratio of EU- and GU-plants. Two reciprocal crossing experiments between EU- and GU-parents resulted in 28.6% and 89.3% EU-individuals, as well as GU-offspring. According to these findings, the qualitative marker ‘proazulene’ (GU-type) is thought to be a recessive character of the tetraploid Achillea species studied.     

Unreduced 3x gamete formation of allotriploid hybrid derived from the cross of Primula denticulata (4x)?×?P. rosea (2x) as a causal factor for producing pentaploid hybrids in the backcross with pollen of tetraploid P. denticulata

A triploid hybrid, which was obtained from interspecific crosses between tetraploid Primula denticulata (2n = 4x = 44) and P. rosea (2n = 2x = 22), successfully produced 11 plants by backcrossing with pollen of tetraploid P. denticulata. Analysis of ploidy level using flow cytometry and chromosome counting in the 11 BC₁ plants revealed that all progeny had much larger DNA contents and chromosome number than both parents. In this triploid-tetraploid (3x–4x) crossing, progeny was predominantly true or near pentaploid presumably produced by the fertilization between true or near triploid female gamete produced from triploid hybrid and diploid pollen of tetraploid P. denticulata. These results suggest that unreduced (3x) or near triploid female gametes were partially produced by single step meiosis, either first-division restitution or second-division restitution process. The zygotes formed by the fertilization between true or near triploid egg produced by single step meiosis in triploid hybrid and diploid pollen produced by normal meiosis of tetraploid P. denticulata might be the only survivors in embryogenesis.     

Induction of haploids and aneuhaploids in colchicine-induced tetraploid Solanum chacoense Bitt.

Summary The species Solanum chacoense BITT. (2n=2x=24) is a tuber-bearing, self-incompatible species which is important both for breeding and for genetic research. It crosses readily with most other tuber-bearing Solanum species including the common potato S. tuberosum (2n=4x=48). Gametophytic incompatibility hampers research in and utilization of this species. Doubling the chromosome number by colchicine makes it a self-compatible autotetraploid. By crossing selfed progeny of 4x-S. chacoense with a number of haploid-inducing diploid Solanum species a high yield of different dihaploid S. chacoense individuals (2n=24) could be obtained from one originally diploid clone: S. chacoense CPC 1153. More than 160 haploids showing a large variability were identified. The average haploid frequency was 53.7 per 100 berries. Most hybrid plants (70–100%) from four 4x × 2x crosses studied were tetraploid. The frequency of triploid hybrids was low (0–10%). Haploid-inducing capacity of fifteen male parents used in this study varied from 0–141 haploids per 100 berries. Careful examination of 156 haploids revealed 15 viable aneuhaploids (2n=25, 26, and 27), i.e. 9.4%. The potential value of these aneuhaploids is discussed.About 50% of the haploids were sufficiently male fertile to use them in crossing. A few of them set berries after selfing. Five aneuhaploids including the one with 27 chromosomes were successfully crossed as females with a diploid hybrid clone.     

The effect of chromosome doubling on fertility,meiotic behaviour and crossability of Solanum etuberosum × S. pinnatisectum

Summary Doubling the chromosome number of sterile F₁ hybrids of S. etuberosum × S. pinnatisectum resulted in fertile allotetraploid hybrids (code 4x-EP) with nearly normal chromosome pairing at meiosis. Many seeds were obtained from selfing and sibbing, but seed set varied considerably. The 4x-EP hybrids were successfully crosses as females with S. stoloniferum and S. polytrichon and as males with S. verrucosum. The crosses with S. stoloniferum and S. polytrichon were carried out reciprocally and unilateral incompatibility was detected. Crosses of 4x-EP hybrids as males onto S. phureja and S. tuberosum cv. Gineke produced parthenocarpic berries only. Nearly 1700 flowers of colchicine-doubled S. pinnatisectum × S. bulbocastanum and the trispecific hybrid (S. acaule × S. bulbocastanum) × S. phureja did not yield a single berry after pollination with a pollen mixture from 20 4x-EP plants.It is concluded that the use of the bridging species S. pinnatisectum and either S. stoloniferum, S. polytrichon or S. verrucosum may enable gene transfer from the non-tuberous Etuberosa species to potato cultivars.     

Pollen donor impact on progenies of pseudogamous blackberries (Rubus subgen. Rubus)

A series of intra- and interspecific crosses in Rubus was evaluated with DNA markers to assess the proportion of apomictically vs. sexually derived progeny plants. The species were well separated from each other with these markers and the interspecific hybrids were easy to ascertain. Ten plants each were derived by selfing of R. hartmanni, R. polyanthemus and R. vestitus, and evaluated with RFLP. Of these plants, only one R. hartmanni plant appeared to be sexually derived, whereas the remainder appeared to have arisen by apomixis. Between 10 and 19 progeny plants, derived by open pollination of R. Hartmanni, R. infestus, R. laciniatus and R. vestitus, respectively (total of 63plants), were evaluated with RFLP. Of these, 6 plants (approx. 10%) showed as lightly deviating band pattern, which may indicate sexual seed set but could also result from experimental artefacts. The three species combinations 1) R. hartmanni and R. sprengelii, 2) R. infestus and R. vestitus, and 3)R. laciniatus and R. polyanthemus were cross-pollinated reciprocally and the resulting progenies evaluated with RAPD (combinations 1 and 2)and leaf shape (combination 3). Combination1 produced 20% (3 out of 15 progenies)sexually derived progenies, combination 2produced 100% (26 progenies) and combination 3 produced 64% (16 out of 26progenies), respectively. The hybrid progenies appeared to have been derived through pollination of unreduced as well as reduced egg cells. The ability to produce interspecific hybrid progenies is most probably dependent upon the species combination. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transfer of high kernel weight and high protein from wild tetraploid wheat (Triticum turgidum dicoccoides) to bread wheat (T. aestivum) using homologous and homoeologous recombination

Summary Wheat pentaploids were produced by hybridizing a high kernel weight (1000 grain wt=56 g), high protein (25.4%) line of wild tetraploid wheat (Triticum turgidum dicoccoides) as male parent, with the three hexaploids (T. aestivum) — normal Chinese Spring and its two homoeologous pairing mutants, ph _1b and ph ₂. The pentaploids were crossed as female parents to the two commercial hexaploid cultivars Warigal and Barkaee and 42-chromosome stable plants selected from the F₁ of the pentaploid x hexaploid crosses.Mean protein content of certain F₃ lines from all six pentaploid x hexaploid crosses was significantly higher than Chinese Spring and the respective commercial hexaploid parent (p<0.005) indicating high protein had been transferred from the tetraploid to the hexaploid level.Kernel weight amongst certain F₃ lines of the three pentaploids x Barkaee was significantly (p<0.0005) higher than either Chinese Spring or Barkaee, indicating the transfer also of high kernel weight from the tetraploid to the hexaploid level. However kernel weight was not significantly increased over Warigal in any F₃ lines of its crosses with the three pentaploids.High levels of homoeologous chromosome pairing in the ph-mutant pentaploids, plus evidence for significant modification of the composition of high-molecular weight (HMW) glutenin subunits of grain protein in certain F₃ derivatives of the ph-mutant pentaploid x hexaploid, crosses indicates that the ph-mutant-derived lines may possess novel (intergenome) genetic recombination, at least for high protein, and possibly kernel weight.     

Intraspecific somatic and sexual hybridisation between dihaploid lines of Solanum tuberosum L.: evaluation of morphological traits and resistance to late blight Phytophthora infestans (Mont.) de Bary in the foliage

Intraspecific tetraploid somatic and sexual hybrid plants have been resynthesised following protoplast fusion and by sexual crosses between two dihaploid potato (Solanum tuberosum) lines each possessing complementary agronomic traits. The dihaploid PDH 40 possesses good tuber shape and yield but has foliage susceptibility to late blight (Phytophthora infestans). On the other hand, the dihaploid PDH 727 possesses resistance to blight in the foliage but has a low yield of small and irregular shaped tubers. Since it was only possible to use a partial selection strategy based on culture media to facilitate recovery of somatic hybrid plants-further morphological and esterase isozyme based characterisations were performed to identify somatic hybrid plants from amongst the non-hybrid plant material. When the blight resistance of both the intraspecific somatic and sexual hybrid plants was assessed there was no significant difference in the mean resistance value and it was intermediate between those of their parents. However, the range of resistance was much wider among the sexual hybrids than among the plants derived from somatic fusion. An assessment of tuber yield between tetraploid sexual and somatic hybrids showed no significant difference and it was higher than that of either parent value. The implication of these results in the context of potato genetics and breeding is discussed.     

Production of non-chimeral colchiploids in Rubus species by tissue culture

Summary Diploid Rubus allegheniensis Porter and R. rusticanus Merc. shoot tips were proliferated in tissue culture and treated with 0.02 to 0.08% colchicine to induce non-chimeral polyploidy. About 20 shoot tips of each treatment were transferred to test tubes containing modified Anderson's medium for shoot proliferation. Shoots at the 3- or 4-leaf stage were transplanted into Jiffy-7 peat pellets and placed under intermittent mist for rooting. Root tip squashes were made for chromosome counts. Thirteen R. allegheniensis and 7 R. rusticanus plants proved to be autotetraploids (28 chromosomes). Stomata of the autotetraploids were significantly larger than those of the diploids but stomatal conductance of CO₂ and photosynthesis rate were reduced significantly as compared to diploids. Pollen grains in tetraploid were larger than those in diploid R. allegheniensis plants. These results indicate that tissue culture can facilitate the production of non-chimeral colchiploids in Rubus spp.     

Parental effects on crossability,embryo differentiation and plantlet recovery in wheat x rye hybrids

Summary One hexaploid wheat cultivar (Triticum aestivem) and two tetraploid wheat lines (T. durum) were crossed with seventeen inbred lines of rye (Secale cereale). Seed set, degree of hybrid embryo differentiation at the time of excision for in vitro culture and recovery of amphihaploid plantlets from various embryo categories were studied. Degree of embryo differentiation was predominantly determined by maternal wheats, paternal rye genotypes appearing to be of minor importance. T. aestivum x rye hybrid embryos were superior to those produced from T. durum for degree of differentiation. The proportion of plantlets developing from differentiated embryos was high for all wheat parents, whereas undifferentiated embryos were mostly unsuitable for plantlet production. The results revealed that cross-incompatibility in hexaploid wheat x rye crosses was due to failure of fertilization, while in tetraploid wheat x rye crosses it was caused by lack of embryo differentiation. Correlation analyses showed that seed set provided a criterion to predict the amphihaploid plantlets to be expected from a particular wheat x rye combination.     

Use of ovule culture to cross between Delphinium species of different ploidy

The applicability of ovule culture was examined in reciprocal crosses between the diploid species D. grandiflorum L. and the tetraploid cultivar D. hybridum ‘Galahad’. An interspecific hybrid plant of D. hybridum ‘Galahad’ × D. grandiflorum was obtained,and hybridity was confirmed by chromosome counting. The hybrid was different from the parents in flower color and size but intermediate between the parents in leaf shape. The hybrid showed low pollen fertility and failed to produce viable seeds by either self- or backcross-pollination using fertile pollen grains from its parents. The applicability and efficiency of the ovule culture technique for genetic improvement of the genus Delphinium through interspecific hybridization between different ploidies is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.